In vitro differentiation of neural stem cells into noradrenergic-like cells.

Neural stem cells (NSCs) as a heterogeneous multipotent and self- renewal population are found in different areas in the developing mammalian nervous system, as well as the sub-ventricular zone (SVZ) and the hippocampus of the adult brain. NSCs can give rise to neurons, astrocytes and oligodendrocytes. The aim of this study was to differentiate neural stem cells into noradrenergic-like cells in vitro. Neural stem cells were harvested from SVZ of newborn rat brains. The cells were cultured in DMEM12, B-27 supplemented with 20 ng/ ml (hFGF) and 20 ng/ ml (EGF) for 2 weeks. Neurospheres were differentiated in neurobasal medium, B-27 supplemented with BDNF (50 ng/ ml) and GDNF (30 ng/ ml) for 3 and 5 days. Cell culture techniques and immunocytochemistry were applied to examine neurospheres and tyrosine hydroxylase positive cells. The number of neurites was counted 3 and 5 days after the induction of differentiation. Nestin and Sox2 were expressed in NSCs and neurospheres. NSCs were differentiated into noradrenergic- like cells (NACs). Tyrosine hydroxylase was detected in these cells. The results of NSCs differentiation for 5 days culture had a significant decrease (P≤0.05) in the number of TH positive cells with one or two neurite per cell, and a significant increase (P≤0.05) in the number of TH positive cells with three, four or more neurites per cell, compared with 3 days culture. Based on these results, NSCs have the ability to differentiate into noradrenergic cells in the presence of BDNF and GDNF growth factors.

eural Stem Cell (NSC) is a multipotent cell which is able to self-renew and proliferate.
NSC resides in a variety of areas in the developing mammalian nervous system as well as in the sub-ventricular zone (SVZ) and the hippocampus of the adult brain (1,2). These cells can generate multiple neural lineages, including neurons, astrocytes and oligodendrocytes (3,4). The SVZ is a region in the brain that is situated throughout the lateral walls of the lateral ventricles. It is a known site of neurogenesis and self-renewing neurons in the adult brain, serving as such due to the interacting cell types, extracellular molecules and localized epigenetic regulation promoting such cellular proliferation (5). Recent in vitro studies of NSC based neurogenesis and gliogenesis have proposed that these processes occur by stepwise limitation and are dependent on environmental signals. Control of NSC proliferation related to the actions of epidermal growth factor (EGF) and/ or its homolog transforming growth factor, basic fibroblast growth factor (FGF-2), may form free floating aggregates termed neurospheres (6)(7)(8). NSC derived neur0000osphere, switch to asymmetric division cycles and give rise to another stem cell and one progenitor cell. The progenitor cells just have the potential to develop into other progenitor cells. Anyway, each clonal neurosphere consists of only a small amount of real stem cells (9). GDNF, RET (receptor tyrosine kinase molecule) and the GDNF co-receptor GFRα1 are expressed by central noradrenergic neurons in regions of the pons, including the A5 and A6 (nucleus locus coeruleus) cell groups (10). GDNF and BDNF are important for the survival, maintenance and regeneration of specific neuronal populations in the adult brain (11,12). GDNF enhances catecholaminergic differentiation of various neuronal cell types in culture (13,14). A5 development culture analysis shows that GDNF affect during early fetal developmental stage to promote the differentiation of noradrenergic neurons without displaying a survival or proliferation change (15). However, GDNF cannot act individually and requires a cofactor like brainderived neurotrophic factor (BDNF) (15).
Previous studies showed that NSCs can be differentiated into neurons and glial cells (3,4). But so far no study has been done on the differentiation of these cells into NACs. Although, an intrinsic neurogenesis and gliogenesis self-repair takes place endogenously during adulthood, NSCs are unable to reconstitute and restore function fully after extensive damage in adult brains. So, the aim of the present study was to evaluate the in vitro differentiation of NSCs into noradrenergic-like cells as a source of adult stem cell for treatment of neurological diseases in future. Trophic factors BDNF (50 ng/ ml; Sigma, UK) and GDNF (30 ng/ ml, Peprotech, UK) were added to the wells for 3 and 5 days (15).

Statistical Analysis
Data have been presented as the mean± SD with at least three biological independent repeats.
Independent sample T-test was used to analyze group differences of the resultant data. The difference between groups was considered as statistically reliable if p≤0.05.

NSCs proliferation
This study demonstrates that in the medium containing hFGF and hEGF, NSCs remain undifferentiated and proliferate extensively. The combination of hEGF and hFGF in the medium enhanced the proliferation of NSCs. Also, the elimination of hEGF and hFGF from medium can differentiate these cells into neurons, astrocytes and oligodendrocytes (Fig. 2 A-I).    Previous studies have confirmed that hEGF and hFGF2 are essential growth factors for proliferation of hNPCs (3,23). The results of our